Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(3): 416-425 (2018)

Enhanced production of alkaline protease from novel

bacterium

Bacillus cereus

GVK21 under submerged

fermentation

Keshavamurthy M

, Vishwanatha T

, Suresh Kumar M

and Subhaschandra M Gaddad*

Department of Post Graduate Studies and Research in Microbiology, Gulbarga University,

Kalaburagi-585106, Karnataka, India

Department of Microbiology, Maharani’s Science College for Women, Bengaluru - 560 001 Karnataka, India

ABSTRACT

Alkaline proteases are an important class of enzymes with potential industrial and commercial applications. In the

present study, 52 bacterial isolates from various soil samples have been evaluated for the production of extracellular

protease and selected one potent isolate based on maximum casein hydrolysis. Further, it is identi ed as Bacillus

cereus GVK21 based on biochemical characteristics and 16S rRNA gene sequence analysis (KY659318). This organism

produced 136 U/mL of protease within 48 hrs of incubation. Maximum production of protease was recorded at pH 9

and a temperature of 40°C. The present study is attempted to exploit new economical media, based on agro wastes

recipes for the increased production of alkaline proteases. B.cereus GVK21 produced high levels of protease (1762 U/

ml) on groundnut oil cake (1.5 %) as a substrate.The results of the study show that this isolate can be further exploited

for commercial production of protease.

KEY WORDS: PROTEASE;

BACILLUS CEREUS

; 16S RRNA; OIL CAKES; INDUSTRIAL PRODUCTION

416

ARTICLE INFORMATION:

*Corresponding Author: smgaddad@gmail.com

Received 12

July, 2018

Accepted after revision 21

Sep, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.3/10

Keshavamurthy et al.

INTRODUCTION

The emergence of new innovations are opening up new

avenues in the areas of industrial biotechnology for the

production of various bulk chemicals and value added

products using inexpensive substrates (Binod et al.,

2013). In the recent years, enzymes are replacing chemi-

cal catalysts in food, leather goods, pharmaceuticals

and textiles industry(Singh etal., 2016). Proteases are a

group of enzymes with wide range of applications, and

account for 40 – 60% of the total enzyme sales with two

thirds of them produced majorly from microorganisms

(Kumar etal., 2011; Deshmukh and Vidhale, 2015).

Proteases are highly complex group of hydrolytic

enzymes and occupy a pivotal status with regard to

their medicinal and industrial applications (El-Bakry

etal., 2015). Proteases are produced by a wide range of

sources(Sharma etal., 2017). The majority of commer-

cially available enzymes are obtained from microbial

origin (Raj etal., 2012). Microbial proteases play a vital

role in biotechnological processes and constitute one of

the most important groups of industrial enzymes, sell-

ing product segment in the global market accounting for

60% market share (Kumar et al., 2014).Bacillus genus

is one of the most important producers of extracellular

proteases and industrial sectors very often use Bacillus

species for the production of proteases(Contesini etal.

2018).

In the recent years, bulk chemicals and value-added

products such as organic acids, amino acids, enzymes,

ethanol, and single cell protein etc., are produced by the

utilization of agro-industrial residues as raw materials

(Pandey etal., 2000; Singhania etal., 2008). Oil cakes

or oil meals are the residues obtained after oil extraction

from the seeds and are rich in proteins,  ber and energy

contents (Ramachandran etal.,2007).The increase in the

demand leads the attention of researchers to explore

novel sources for proteases, where isolation, screening

and characterization of new promising strains are a con-

tinuous process. Therefore, the present study focuses on

the selection of high yielding stable proteolytic bacte-

ria from mining soil sample which is considered as an

extreme environment region.

MATERIALS AND METHODS

SAMPLE COLLECTION AND ISOLATION

Soil samples were collected in sterile containers from

different locations within the mining area of Raichur

district (16

’

”

North Latitude and 76

38’ 31

”

East

Longitude), Karnataka, India. One gram of soil was

added into100 mL of enrichment medium [g/L: Casein –

10.0 and NaCl – 5.0 at pH 9.0] in a 250 mL of Erlenmeyer

 ask and incubated at 37

C in a rotary shaker incuba-

tor at 140 rpm for 24 hours. A loopful of the enriched

medium was streaked on Nutrient agar (NA) plates and

incubated at 37

C for 24 hours. Well isolated colonies

were re-streaked on NA for con rming the purity and

transferred onto NA slants and preserved for further use.

SCREENING OF THE ISOLATES FOR

EXTRACELLULAR PROTEASE PRODUCTION

The isolates were screened for protease production on

screening medium, skimmed milk agar [g/L: skim milk -

10.0; peptone - 5.0; yeast extract - 3.0; NaCl - 5.0; agar,

20 and pH 9.0](Kumar etal., 2011). After 24 hrs growth,

the plates were observed for clear zones around the colo-

nies. Proteolytic activity was further con rmed by using

gelatin as the substrate in the medium (Pant etal.,2015).

Depending on the zone of clearance,  ve isolates GVK8,

GVK21, GVK29, GVK38 and GVK40, were selected for

further experimental studies.

PROTEASE PRODUCTION UNDER SUBMERGED

FERMENTATION

Protease production by the selected isolates was carried

out by submerged fermentation. One mL of fresh bacte-

rial inoculum was added in to 250 mL Erlenmeyer  ask

containing 100 mL of production medium [g/L: casein-

5.0, yeast extract - 5.0; NaCl - 5.0; MgSO

O - 0.02

and KH

- 0.05 at pH 9.0]. The  asks were placed in

a rotary shaker incubator at 140 rpm at 37° C for 3-4

days. An aliquot of the culture supernatant was collected

at regular time intervals of 12 hrs and assayedfor protease

activity (Josephine etal.,2012). Based on the maximum

protease production and shorter time required, the isolate

GVK 21 is a potent strain and selected for further studies

IDENTIFICATION OF THE POTENT PROTEASE

PRODUCING ISOLATE

Morphological and biochemical characteristics of the

selected isolate (GVK21) was studied and recorded as

per Bergey’s Manual of Systematic Bacteriology (Holt

etal., 2000). The bacterial isolate was further identi ed

by 16S rDNA sequence analysis using universal primers

and genomic DNA as template. The genomic DNA of the

isolate was extracted as described by Roohi etal., (2012).

The PCR ampli ed product was sequenced at Microbial

Ecology Laboratory, National Centre for Cell Science,

Pune. Phylogenetic tree was constructed with Molecu-

lar Evolutionary Genetics Analysis (MEGA) 6.0 software

using neighbor-joining method (Tamura et al.,2011).

Duly annotated partial nucleotide sequences of the

novel bacterial strain was deposited with NCBI Genbank

(http://www.ncbi.nlm.nih.gov).

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ENHANCED PRODUCTION OF ALKALINE PROTEASE 417

Keshavamurthy et al.

SCANNING ELECTRON MICROSCOPY

Scanning Electron Microscopic (SEM) analysis was done

to observe the morphology of the isolated strain.Thin

 lm of the sample was prepared on a carbon coated cop-

per grid by just dropping a small amount of the bacterial

culture on the grid. Extra solution was removed using

a blotting paper. Then the  lm on the SEM grid was

allowed to air dry by putting it under a mercury lamp

for 5 min. The sample was then observed under scanning

electron microscopy TESCAN (Vega 3 LMU) at a resolu-

tion of 3 nm at various magni cations at acceleration

voltage of 20.0 KV.

PROTEASE PRODUCTION IN RELATION TO THE

GROWTH OF BACTERIA

In order to study the time course for microbial growth

and protease production, the isolate B. cereus GVK 21

was inoculated in the production medium and incubated

in rotary shaker incubator at 160 rpm at 40° C upto 72

hours. The growth of the bacterium was determined by turbi-

dometry, OD at 600 nm. After the removal of cells by centrifu-

gation, the cell free supernatant was considered as the crude

enzyme solution and protease activity was measured.

EVALUATION OF AGRO RESIDUES (OIL CAKES)

FOR ENHANCED PROTEASE PRODUCTION

Major regional Oil cakes such as neem and pongamia

were collected from local market, Bengaluru, Indiaand

were  ne powdered using mixer grinderand evaluated

as substrates for the production of protease. Different

type of oil cakes such as Castor oil cake (COC), Ground-

nut oil cake (GOC), Neem oil cake (NOC) and Pongamia

oil cake (POC) were supplemented individually in to 100

mL of the optimized production mediumin 250 mL of

Erlenmeyer  ask. The agro wastes were used at a con-

centration of 0.5 to 2% with an increment of 0.25 %. The

amount of protease produced was determined at every 6

h up to 72 h.

PROTEASE ASSAY

The culture was centrifuged at 10,000 rpm for 5 min

at 4° C to obtain the CFS. The protease activity of the

crude enzyme was determined by the modi ed method

of Joo et al.,(2002) brie y; 0.5 mL of CFS was added

to 0.5 mL of 1% casein (as a substrate) in 0.1 M Tris-

HCl (pH 9.0) and incubated at room temperature for 10

min. The reaction was terminated by the addition of 3

mL of 10% (w/v) trichloroacetic acid (TCA). The solution

was centrifuged at 5000 rpm for 10 min. To the 3 mL

of the clear supernatant, 5 mL 0.4 M sodium bicarbo-

nate solution and 0.5 mL of Folin Ciocalteau reagent

(FCR) were added, mixed thoroughly and incubated for

30 min at room temperature, in dark. The optical den-

sity was measured using a UV-VIS spectrophotometer

(ELICO SL-159) at 660 nm against the enzyme blank.

The amount of the released aromatic amino acids was

calculated usingtyrosine standard.

One unit of protease is de ned as the amount of the

enzyme required to release 1g of tyrosine per mL per

min under the above assay conditions. Enzyme activity

was calculated according to the formula of Pant etal.,

(2015).

RESULTS AND DISCUSSION

ISOLATION, IDENTIFICATION, SCREENING

AND CHARACTERIZATION OF PROTEASE-

PRODUCING BACTERIA

In the present study, 52 independent bacteria were iso-

lated from the soil samples collected from gold mines

and were screened for proteolytic activity on skim milk

agar plates (Figure 1). A total of 28 (53.84 %) isolates

showed proteolytic activity ranging from 11 - 37 mm

of clear zones aroundthe colonies. Five bacterial iso-

lates, GVK8, GVK21, GVK29, GVK38 and GVK40, were

selected based on the higher zone of hydrolysis on skim

milk agar plates for further study. The use of skim milk

agar medium for the ef cient screening of proteolytic

bacteria has been reported by earlier researchers (Raj

etal., 2012; Ravi etal., 2015).

The selected  ve isolates were further screened for

the quantitative production of the enzyme in production

medium (Figure 2). The protease production was slower

during the  rst 12 hours by all the isolates. The protease

production increased signi cantly in respect of the iso-

late GVK21 during 12 to 24 hours to reach the maximum

activity and remained at that level till 48 hours. While

with respect to the other isolates, the protease activity

increased gradually and the maximum activities were

recorded at 48 hours of incubation. This indicated that

the isolate GVK21 produces a maximum protease activity

of 136 U/mL and takes shorter time in preliminary study.

The strain GVK21 is gram positive rod, motile,

spore former and was tentatively identi ed as Bacillus

sp. based on its morphological and biochemical char-

acteristics (Table 1), (Holt et al.,2000). The 16S rDNA

was ampli ed through PCR which showed1500 kb band

on 2% agarose gel (Figure 3). Subsequently, the com-

parison of the 16S rRNA gene nucleotide sequence (1387

bp) of the strain GVK21 with other 16S rRNA genes

sequences of closely related strains from NCBI database

showed that this isolate has 99 % sequence homology

with B. cereus ATCC 14579(Accession No. 074540). The

418 ENHANCED PRODUCTION OF ALKALINE PROTEASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Keshavamurthy et al.

FIGURE 1. Zone of hydrolysis on A) skim milk agar B) Gelatin agar by bacterial isolate Bacillus cereus

GVK 21.

FIGURE 2. Quantitative production of protease using different bacterial isolates.

phylogenetic tree, constructed by the neighbor-joining

method indicated that the strain GVK21 is af liated

with the genus Bacillus and closely related to B. cereus

strain LG1 - Accession No. KF307764 (Figure 4). The

obtained nucleotide sequence GVK21 was submitted to

GenBank database and the accession number assigned

is KY659318 (https://www.ncbi.nlm.nih.gov/ nuccore/

KY659318). The species B. cereusATCC 14579(074540.1),

B. cereus strain JCM 2152 (113266.1), B. thuringiensis

strain NBRC 101235 (112780.1) has the closest sequence

similarity of 99%.

Bacillus is an industrially important organism and

was the  rst to be used in the commercial production

of protease in 1952 (Binod et al., 2013). Several pro-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ENHANCED PRODUCTION OF ALKALINE PROTEASE 419

Keshavamurthy et al.

teases have been produced from many Bacillus species

(Rao et. al., 1998). Bacillus species like B. subtilis (Babe

and Schmidt, 1998), B. licheniformis (Mabrouk et al.,

1999), B. sphaericus (Singh etal., 1999), B. proteolyti-

cus (Bhaskaretal., 2007), Bacillus cereus (Doddapaneni

etal., 2009; Bajaj etal., 2013) and B. megaterium (Raj-

kumaretal., 2010) have been reported for protease pro-

duction. Other than Bacillus species bacteria such as

Pseudomonas  uorescens (Kalaiarasi and Sunitha, 2009),

P. aeruginosa (Raj etal., 2012), Vibrio etschnikovii (Jel-

loulietal., 2009), V. alginolyticus (Shanthakumari etal.,

2010) are also reported for protease production.

Bacillus cereus GVK 21 was subjected for secondary

screening for quantitative protease production under

submerged condition. The protease production increased

signi cantly from 12 U/mL to 136 U/mL during6 h to

36 h of incubation and the enzyme activity was in syn-

chrony with the growth of the bacterium, wherein the

logarithmic growth was observed during 6 to 24 hours

and the maximum growth was observed at 36 hours

Table 1. Morphological and biochemical characteristics

of B. cereus strain GVK21 isolated from soil.

Morphological Characteristics Results

Gram staining Positive rods

Colour Creamish white

Motility test Motile

Spore Spore former

Physiological characteristics

Catalase Positive

Indole Negative

Methyl red Negative

Voges Proskauer Positive

Citrate utilization Positive

Oxidase reaction Positive

Casein hydrolysis Positive

Gelatin liquefaction Positive

Starch hydrolysis Positive

Nitrate reduction Positive

Growth at 4o C -

Growth at 40o C +

FIGURE 3. Agarose Gel Electrophoresis of

B.cereus DNA. Lane1- 100 bp DNA lad-

der, Lane2- 16S rDNA amplicon of B.cereus

GVK21

FIGURE 4. Phylogram obtained based on phylogenetic analysis of 16S rDNA gene sequence data showing the phyloge-

netic positions of isolate Bacillus cereus strain GVK21 and of a number of related taxa

420 ENHANCED PRODUCTION OF ALKALINE PROTEASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Keshavamurthy et al.

FIGURE 5. Protease production with relation to growth of Bacillus cereus GVK 21.

FIGURE 6. SEM images of B.cereus GVK 21

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ENHANCED PRODUCTION OF ALKALINE PROTEASE 421

Keshavamurthy et al.

FIGURE 7. Different Oil cakes used as a substrate for protease production.

indicating that the protease production is maximum in

the late exponential and early stationary phase (Figure

5). The decline in the protease production was observed

after 48 h. Kannikar et al.,(2008) reported that Bacil-

lus sp. BA40 produced 1.158 U/mL protease activity, B.

licheniformis LBBL-11 showed 18.4 U/mL at 48 hours

(Olajuyigbe, and Ajele,2008). The results indicate that

the isolate B. cereus GVK 21 is producing 136U/mL a

comparatively higher protease activity, that too under

preliminary screening.

Chitte and Dey (2000) have also shown that the log

phase the optimum for the production ofprotease by

Streptomyces megasporus. Incubation time of 24 hours

positively in uences the production of protease from

Bacillus cereus SRM-001 (Narasimhan et al.,2015).

Incubation time of 40 h was found to enhance protease

production by Bacillus natto-NRRL-3666 (Mahajan

etal.,2010).

SEM ANALYSIS

Figure 6 shows SEM image of bacteria which con rmed

the rod shaped nature of the cells, where the cell size

(length and breadth) is found to be 1.91 µm and 0.82 µm

respectively.

ENHANCED PROTEASE PRODUCTION USING

OIL CAKES

The effect of agro-based by products as alternative

substrates for bacterial protease production under sub-

merged fermentation has been studied by several work-

ers (Praveen Kumar et al., 2008, Prasad, et al., 2014).

B. cereus GVK21 (KY659318) produced varying levels

of alkaline protease on various agriculture based sub-

strates. Among various oil cakes (Figure. 7) examined,

groundnut oil cake enhanced the protease production

from B. cereus GVK21 by 112 % (1762 U/ml) as com-

pared to control (665 U/ml), i.e., groundnut oil cake

supported maximum protease production. Bacillus sub-

tilis SHS-04exhibited maximum protease production

(1616.21 U/mL) by utilizing groundnut cake as substrate

(Olajuyigbe, 2013).

Other oil cakes also substantially supported protease

production, castor cake (1316 U/ml), pongamia cake

(1144 U/ ml) and neem cake (904 U/ ml) as shown in

Figure 8. Enzyme synthesis was found to be repressed

by rapidly metabolizable nitrogen sources such as amino

acids or ammonium ion concentrations in the medium

as also observed in the presence of ammonium sulphate

and potassium nitrate (Saurabh etal., 2007; Bajaj and

422 ENHANCED PRODUCTION OF ALKALINE PROTEASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Keshavamurthy et al.

Sharma, 2011). Therefore complex nitrogen sources

are usually used for protease production. Groundnut

oil cake, a rich protein source (30–40%), which mainly

constitutes different amino acids such as arginine (11.0),

Leucine (6.1), glycine (6.0), and phenyl alanine (4.9)

and a dry matter of 92.6% comprehends slow release of

nitrogen due to its conditioned and moderately complex

nature which may favor the process organism metaboli-

cally and physiologically for ef cient production of pro-

tease (Kuo, 1967). Several reports indicate that ground-

nut cake serves as a reasonably good nitrogen source

for protease production by various microorganisms

(Kuberan etal., 2010; Kranthi etal., 2013, Olajuyigbe,

2013).

The substrate in the growth medium constitutes a

major cost determining factor for the commercial pro-

duction of industrial enzymes. The high cost of protease

production is another major hindrance for wide range

of industrial and medicinal applications of this enzyme

(Jayasree etal., 2009). Utilization of agro industrial resi-

dues as carbon and nitrogen sources for the bulk pro-

duction of industrial enzymes may play a signi cant

role not only in reducing the production cost and also

contribute towards growing environmental concerns by

addressing the agro industrial waste disposal and man-

agement problems because of tremendous quantities

of agricultural residues generated through agricultural

practices and industrial processes (Bajaj and Wani, 2011;

Singh and Bajaj 2015). Earlier workers also reported that

defatted oil meals like soybean meal (Saurabh et al.,

2007), cotton seed cake (Bajaj etal., 2013) and ground-

nut meals (Olajuyigbe, 2013) are the cost-effective alter-

natives for industrial production processes. Hence utili-

zation of a low-priced nitrogen source is an important

criterion for economic production of industrial enzymes.

Requirement of speci c nitrogen source differs from one

organism to other even among the same species isolated

from different sources (Bajaj and Sharma, 2011).

CONCLUSION

Bacillus cereus GVK 21 (KY659318) isolated from the soil

samples from a mining area exhibited higher proteolytic

activity of 136 U/mL which signi cantly increased to

1762 U/mL on ground nut cake, comparatively higher

than already reported and thus can be exploited as a

potential source for large scale production of protease

enzyme to cope up the needs of industrial applications

and the demand of the global market.

FIGURE 8. Enhanced Protease production using different oil cakes.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ENHANCED PRODUCTION OF ALKALINE PROTEASE 423

Keshavamurthy et al.

ACKNOWLEDGMENTS

Authors wish to acknowledge Dr. Yogesh Souche, Scien-

tist, National Centre for Cell Science, Govt of India, Pune

for sequencing and identi cation of Bacteria and B.M.S

College of Engineering, Bengaluru, Karnataka, India for

SEM analysis. Authors are thankful to the Maharani’s

Science College for Women, Bengaluru, Karnataka, India

for providing facilities to carry out this work.

CONFLICT OF INTEREST

Authors declare that they have no con ict of interest in

the publication.

REFERENCES

Babe, L., and Schmidt, B., 1998. Puri cation and biochemi-

cal analysis of WprA, a 52-kDa serine protease secreted by B.

subtilis as an active complex with its 23-kDa propeptide.Bio-

chimicaet Biophysica Acta (BBA)-Protein Structure and Molec-

ular Enzymology,1386(1),pp.211-219.

Bajaj, B.K. and Sharma, P., 2011. An alkali-thermotolerant

extracellular protease from a newly isolated Streptomyces sp.

DP2.New biotechnology,28(6), pp.725-732.

Bajaj, B.K. and Wani, M.A., 2011. Enhanced phytase production

from Nocardia sp. MB 36 using agroresidues as substrates:

Potential application for animal feed production.Engineering

in Life Sciences,11(6), pp.620-628.

Bajaj, B.K., Sharma, N. and Singh, S., 2013. Enhanced produc-

tion of  brinolytic protease from Bacillus cereus NS-2 using

cotton seed cake as nitrogen source.Biocatalysis and Agricul-

tural Biotechnology,2(3), pp.204-209.

Bhaskar, N., Sudeepa, E.S., Rashmi, H.N. and Selvi, A.T., 2007.

Partial puri cation and characterization of protease of Bacil-

lus proteolyticus CFR3001 isolated from  sh processing waste

and its antibacterial activities.Bioresource Technology,98(14),

pp.2758-2764.

Binod, P., Palkhiwala, P., Gaikaiwari, R., Nampoothiri, K.M.,

Duggal, A., Dey, K. and Pandey, A., 2013. Industrial Enzymes-

Present status and future perspectives for India.

Chitte, R.R. and Dey, S., 2000. Potent  brinolytic enzyme from

a thermophilic Streptomyces megasporusstrain SD5.Letters in

applied microbiology,31(6), pp.405-410.

Contesini, F.J., Melo, R.R.D. and Sato, H.H., 2018. An overview

of Bacillus proteases: from production to application.Critical

reviews in biotechnology,38(3), pp.321-334.

Doddapaneni, K.K., Tatineni, R., Vellanki, R.N., Rachcha,

S., Anabrolu, N., Narakuti, V. and Mangamoori, L.N., 2009.

Puri cation and characterization of a solvent and detergent-

stable novel protease from Bacillus cereus. Microbiological

Research,164(4), pp.383-390.

El-Bakry, M., Abraham, J., Cerda, A., Barrena, R., Ponsá, S.,

Gea, T. and Sánchez, A., 2015. From wastes to high value

added products: novel aspects of SSF in the production of

enzymes.Critical Reviews in Environmental Science and Tech-

nology,45(18), pp.1999-2042.

Holt, J.G., Krieg, N.R., Sneath, P., Staley, J.T. and Williams, S.T.,

Bergey’s Manual of Determinative Bacteriology. 2000.Lippin-

cot Williams & Wilkins. pag, pp.571-572.

Jayasree, D., Kumari, T.D.S., Kishor, P.B.K., Lakshmi, M.V. and

Narasu, M.L., 2009. Optimization of production protocol of

alkaline protease by.Streptomyces pulvereceus; Inter JRI Sci-

ence and Technology,1(2).

Jellouli, K., Bougatef, A., Manni, L., Agrebi, R., Siala, R.,

Younes, I. and Nasri, M., 2009. Molecular and biochemi-

cal characterization of an extracellular serine-protease from

Vibrio metschnikovii J1.Journal of industrial microbiology &

biotechnology,36(7), pp.939-948.

Joo, H.S., Kumar, C.G., Park, G.C., Kim, K.T., Paik, S.R. and

Chang, C.S., 2002. Optimization of the production of an extra-

cellular alkaline protease from Bacillus horikoshii. Process

Biochemistry,38(2), pp.155-159.

Josephine, F.S., Ramya, V.S., Devi, N., Ganapa, S.B. and Vishwa-

natha, T., 2017. Isolation, production and characterization of

protease from Bacillus sp isolated from soil sample. Journal

of Microbiology and Biotechnology Research, 2(1), pp.163-

168.

Kalaiarasi, K. and Sunitha, P.U., 2009. Optimization of alkaline

protease production from Pseudomonas  uorescens isolated

from meat waste contaminated soil. African Journal of Bio-

technology,8(24).

Kranthi, V.S., Rao, D.M. and Jaganmohan, P., 2012. Protease

production by Rhizopus stolonifer through solid state fermen-

tation.Cent. Euro. J. Exp. Bio,1, pp.113-117.

Kumar, E.V., Srijana, M., Kumar, K.K., Harikrishna, N. and

Reddy, G., 2011. A novel serine alkaline protease from Bacillus

altitudinis GVC11 and its application as a dehairing agent.Bio-

process and Biosystems engineering,34(4), pp.403-409.

Kumar, D., Kumar, V., Verma, A.K. and Dubey, A., 2013. Kinetic

characterization and immobilization of partially puri ed

extracellular alkaline protease from rhizospheric soil bacte-

rium Bacillus subtilis strain EN4.J. Pure Appl. Microbiol,7(1),

pp.727-732.

Kumar, P.P., Mathivanan, V., Karunakaran, M., Renganathan,

S. and Sreenivasan, R.S., 2008. Studies on the effects of pH

and incubation period on protease production by Bacillus spp.

using groundnut cake and wheat bran.Indian Journal of Sci-

ence and Technology,1(4), pp.1-4.

Mabrouk, S.S., Hashem, A.M., El-Shayeb, N.M.A., Ismail, A.M.

and Abdel-Fattah, A.F., 1999. Optimization of alkaline protease

productivity by Bacillus licheniformis ATCC 21415. Biore-

source Technology,69(2), pp.155-159.

Maghsoodi, V., Kazemi, A., Nahid, P., Yaghmaei, S. and

Sabzevari, M.A., 2013. Alkaline protease production by immo-

bilized cells using B. licheniformis. Scientia Iranica, 20(3),

pp.607-610.

Mahajan, P.M., Gokhale, S.V. and Lele, S.S., 2010. Production

of nattokinase using Bacillus natto NRRL 3666: media opti-

424 ENHANCED PRODUCTION OF ALKALINE PROTEASE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Keshavamurthy et al.

mization, scale up, and kinetic modeling. Food Science and

Biotechnology,19(6), pp.1593-1603.

Narasimhan, M.K., Chandrasekaran, M. and Rajesh, M., 2015.

Fibrinolytic enzyme production by newly isolated Bacillus

cereus SRM-001 with enhanced in-vitro blood clot lysis poten-

tial.The Journal of general and applied microbiology,61(5),

pp.157-164.

Olajuyigbe, F.M. and Ajele, J.O., 2008. Some properties of

extracellular protease from Bacillus licheniformis LBBL-11 iso-

lated from iru, a traditionally fermented African locust bean

condiment. African Journal of Biochemistry Research, 2(10),

pp.206-210.

Olajuyigbe, F.M., 2013. Optimized production and properties of

thermostable alkaline protease from Bacillus subtilis SHS-04

grown on groundnut (Arachis hypogaea) meal. Advances in

Enzyme Research,1(04), p.112.

Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., Vandenberghe,

L.P. and Mohan, R., 2000. Biotechnological potential of agro-

industrial residues. II: cassava bagasse. Bioresource technol-

ogy,74(1), pp.81-87.

Pant, G., Prakash, A., Pavani, J.V.P., Bera, S., Deviram, G.V.N.S.,

Kumar, A., Panchpuri, M. and Prasuna, R.G., 2015. Production,

optimization and partial puri cation of protease from Bacillus

subtilis.Journal of Taibah University for Science,9(1), pp.50-55.

Prasad, R., Abraham, T.K. and Nair, A.J., 2014. Scale up of

production in a bioreactor of a halotolerant protease from

moderately halophilic Bacillus sp. isolated from soil.Brazilian

Archives of Biology and Technology,57(3), pp.448-455.

Raj, A., Khess, N., Pujari, N., Bhattacharya, S., Das, A. and

Rajan, S.S., 2012. Enhancement of protease production by

Pseudomonas aeruginosa isolated from dairy ef uent sludge

and determination of its  brinolytic potential. Asian Paci c

Journal of Tropical Biomedicine,2(3), pp.S1845-S1851.

Rajkumar, R., Jayappriyan, K.R., Kannan, P.R. and Rengasamy,

R., 2010. Optimization of Culture Conditions for the Production

of Protease from Bacillus megaterium.Journal of Ecobiotech-

nology.

Rao, M.B., Tanksale, A.M., Ghatge, M.S. and Deshpande, V.V.,

1998. Molecular and biotechnological aspects of microbial

proteases.Microbiology and molecular biology reviews,62(3),

pp.597-635.

Ramachandran, S., Singh, S.K., Larroche, C., Soccol, C.R. and

Pandey, A., 2007. Oil cakes and their biotechnological appli-

cations–A review. Bioresource technology, 98(10), pp.2000-

2009.

Ravi, M., Rayudu, K., Gaddad, S.M. and Jayaraj, Y.M., 2015.

Studies on the potent protease producing bacteria from soil

samples.Int. J. Curr. Microbiol. App. Sci,4(1), pp.983-988.

Rupali R. Deshmukh and Vidhale N N.,2015. Effect of pH on

the production of protease by Fusarium oxysporum using

agroindustrial waste. Biosci. Biotech. Res. Comm. 8(1): 78-83

(2015)

Santong, K., Chunglok, W., Lertcanawanichakul, M. and Ban-

grak, P., 2011. Screening and isolation of Bacillus sp. produc-

ing thermotolerant protease from raw milk.Walailak Journal

of Science and Technology (WJST),5(2), pp.151-160.

Saurabh, S., Jasmine, I., Pritesh, G. and Kumar, S.R., 2007.

Enhanced productivity of serine alkaline protease by Bacillus

sp. using soybean as substrate.Malaysian Journal of Microbi-

ology,3(1), pp.1-6.

Shanthakumari, A.R., Nagalakshmi, R. and Ramesh, S., 2010.

Scaleup and media optimization of protease by Vibrio algino-

lyticus.Journal of Ecobiotechnology.

Sharma, K.M., Kumar, R., Panwar, S. and Kumar, A., 2017.

Microbial alkaline proteases: Optimization of production

parameters and their properties.Journal of Genetic Engineer-

ing and Biotechnology,15(1), pp.115-126.

Singh, J., Vohra, R.M. and Sahoo, D.K., 1999. Alkaline protease

from a new obligate alkalophilic isolate of Bacillus sphaeri-

cus.Biotechnology letters,21(10), pp.921-924.

Singh, S. and Bajaj, B.K., 2015. Medium optimization for

enhanced production of protease with industrially desirable

attributes from Bacillus subtilis K-1. Chemical Engineering

Communications,202(8), pp.1051-1060.

Singh, R., Kumar, M., Mittal, A. and Mehta, P.K., 2016. Micro-

bial enzymes: industrial progress in 21st century. 3 Bio-

tech,6(2), p.174.

Singhania, R.R., Soccol, C.R. and Pandey, A., 2008. Application

of tropical agro-industrial residues as substrate for solid-state

fermentation processes. InCurrent developments in solid-state

fermentation(pp. 412-442). Springer, New York, NY.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M.

and Kumar, S., 2011. MEGA5: molecular evolutionary genet-

ics analysis using maximum likelihood, evolutionary distance,

and maximum parsimony methods. Molecular biology and

evolution,28(10), pp.2731-2739.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ENHANCED PRODUCTION OF ALKALINE PROTEASE 425